Detection of negatively charged polymers using water-soluble, cationic, polythiophene derivatives

ABSTRACT

Novel methods allowing for the simple optical and electrochemical detection of double-stranded oligonucleotides are disclosed. The methods are rapid, selective and versatile. Advantageously, they do not require any chemical reaction on the probes or on the analytes since they are based on different electrostatic interactions between cationic poly(3-alkoxy-4-methylthiophene) derivatives and single-stranded or double-stranded (hybridized) oligonucleotides.

RELATED APPLICATIONS

The present application is a divisional of U.S. Ser. No. 11/370,158,filed on Mar. 7, 2006, now U.S. Pat. No. 7,446,213 by Leclerc et al.,and entitled “DETECTION OF NEGATIVELY CHARGED POLYMERS USINGWATER-SOLUBLE, CATIONIC, POLYTHIOPHENE DERIVATIVES,” which is adivisional U.S. Ser. No. 10/474,230, filed on Apr. 5, 2004, by Leclerc,et al., and entitled “DETECTION OF NEGATIVELY CHARGED POLYMERS USINGWATER-SOLUBLE, CATIONIC, POLYTHIOPHENE DERIVATIVES,” issued as U.S. Pat.No. 7,083,928, which claims priority under 35 U.S.C. § 119(a)PCT/CA02/00485 filed Apr. 5, 2002, entitled “DETECTION OF NEGATIVELYCHARGED POLYMERS USING WATER-SOLUBLE, CATIONIC, POLYTHIOPHENEDERIVATIVES,” which claims priority to U.S. Provisional Application No.60/281,371, filed Apr. 5, 2001; U.S. Provisional Application No.60/284,184 filed Apr. 18, 2001; and U.S. Provisional Application No.60/288,442 filed May 4, 2001, each of which is hereby expresslyincorporated in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a sequence listing inelectronic format. The sequence listing is provided as a file entitledGENOM.052NPDD.txt, created Oct. 7, 2008 which is 3.25 KB in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to simple and reliable methods fornegatively charged polymer detection, namely for sequence-selectivenucleic acid detection. More specifically, the present invention relatesto sequence-selective nucleic acid detection methods, which areessential for the rapid diagnosis of infections and a variety ofdiseases.

2. Description of the Related Art

Complexes of polythiophene derivatives bearing sulfonic acid moietiesand one or several adequately designed amine-containing molecules(electrostatic interactions) have been shown to be responsive toexternal stimuli (PCT/CA98/01082). More specifically, they were shown toundergo striking conformational changes when exposed to heat, light orvarious chemical and biochemical moieties giving rise to thermochromism,photochromism, ionochromism or even biochromism. These sulfonicacid-bearing polythiophene derivatives are not positively charged andthus do not have any particular affinity for negatively chargedpolymers.

The search for methods for sequence-selective nucleic acid detection hasevolved into an important research field and has subsequently drawn theattention of researchers from various disciplines such as chemistry,physics, biochemistry, etc. As a result, some interesting DNAhybridization sensors have recently been proposed. (Fodor, S. P. A etal. Science 251, 767, (1991); Livache, T. et al. Nucleic Acids Res. 22,2912 (1994); Tyagi, S.; Kramer, F. R. Nature Biotechnology 14, 303(1996); Mikkelson, S. R. Electroanalysis 8, 15 (1996); Taton, T. A.;Mirkin, C. A.; Letsinger, R. L. Science 289, 1757, (2000)).

However, most of these newly developed approaches perform detection byattaching a fluorescent or electro-active tag to the analyte.

Assays that do not require nucleic acid functionalization prior todetection are of greater fidelity and several research groups havereported the utilization of conjugated field-responsive polymers(polypyrroles, polythiophenes, etc.) as electrochemical or opticaltransducers. (Leclerc, M. Adv. Mater. 11, 1491, (1999); McQuade, D. T.;Pullen, A. E.; Swager, T. M. Chem. Rev. 100, 2537 (2000); Chen, L.,McBranch, D. W., Wang, H. L., Hegelson, R., Wudl, F. & Whitten, D. G.Highly sensitive biological and chemical sensors based on reversiblefluorescence quenching in a conjugated polymer. PNAS 96, 12287 (1999);Ewbank, P. C., Nuding, G., Suenaga, H., McCullough, R. D., Shinkai, S.Amine functionalized polythiophenes: synthesis and formation of chiral,ordered structures on DNA substrates. Tetrahedron Lett. 42, 155 (2001)).Indeed, the ability of some oligonucleotide-functionalized conjugatedpolymers to transduce hybridization events into an electrical or opticalsignal, without utilizing any labeling of the analyte, has beendemonstrated. (Youssoufi, H. K.; Garnier, F.; Srivastava, P.; Godillot,P.; Yassar, A. J. Am. Chem. Soc., 119, 7388, (1997); Bauerle, P.; Emge,A. Adv. Matter. 10, 324, (1998); Garnier, F.; Youssoufi, H. K.;Srivastava, P.; Mandrand, B.; Delair, T. Synth. Metals, 100, 89,(1999)). The detection mechanism is based on a modification of theelectrical and/or optical properties through the capture of thecomplementary oligonucleotides.

There thus remains a need for simpler, more sensitive and more reliablemethods for the rapid and specific identification of nucleic acids.These nucleic acids could be used for the diagnosis of infections anddisease. Ideally, an assay that does not require nucleic acidfunctionalization (chemical manipulation of nucleic acids) prior todetection nor complex reaction mixtures would have the followingcharacteristics: it would be simpler to use than the assays currentlyavailable and it would have a high degree of fidelity. Such an assaywould be highly beneficial and therefore very desirable.

The present invention seeks to meet these and other needs.

SUMMARY OF THE INVENTION

In general terms, the present invention relates to novel cationic,water-soluble polythiophene derivatives, which can readily transduceoligonucleotide hybridization into a clearly interpretable optical(calorimetric, fluorescent or luminescent) or electrical signal. Thesepolymers can discriminate between specific and non-specifichybridization of nucleic acids differing by only a single nucleotide.

Specifically, the present invention relates to the synthesis and use ofcationic water-soluble polymers composed of thiophene monomers havingthe following general formula:

wherein “m” is an integer ranging from 2 to 3; R* is a quaternaryammonium; Y is an oxygen atom or a methylene; and R¹ is a methyl groupor a hydrogen atom.

The present invention further comprises a method for detecting thepresence of negatively charged polymers, comprising the steps of:

-   -   a) contacting a complementary target to the negatively-charged        polymer with a thiophene polymer to form a duplex;    -   b) contacting the duplex with the negatively charged polymer;        and    -   c) detecting a change in electronic charge, fluorescence or        color as an indication of the presence of the negatively charged        polymer.

The negatively charged polymers for the method as described above, maybe selected from the group consisting of: acidic proteins,glycosaminoglycans, hyaluronans, heparin, chromatographic substrates,culture substrates and nucleic acids.

The present invention additionally comprises a method for discriminatinga first nucleic acid from a second nucleic acid, the second nucleic aciddiffering from the first nucleic acid by at least one nucleotide,comprising the steps of:

-   -   a) contacting a complementary target to the first nucleic acid        with a thiophene polymer to form a duplex;    -   b) contacting the duplex with the first nucleic acid, resulting        in specific hybridization;    -   c) contacting the duplex with the second nucleic acid, resulting        in non-specific hybridization; and    -   d) detecting a change in electronic charge, fluorescence or        color

Finally, the present invention contemplates a number of specificapplications, such as the use of a positively charged polymer comprisinga repeating thiophene moiety for detecting the presence of negativelycharged polymers, and for purifying negatively charged polymers.

Further scope and applicability will become apparent from the detaileddescription given hereinafter. It should be understood however, thatthis detailed description, while indicating preferred embodiments of theinvention, is given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the UV-visible absorption spectrum of a 2.4×10⁻⁵ M (on amonomeric unit basis) solution of: a) polymer 2; b) polymer 2/X1 duplex;c) polymer 2/X1/Y1 triplex, at 55° C. in 0.1M NaCl/H₂O using an opticalpath length of 1 cm.

FIG. 2 shows the UV-visible absorption spectrum of a 2.4×10⁻⁵ M (on amonomeric unit basis) solution of: a) polymer 2; b) polymer 2/X1 duplex;c) polymer 2/X1/X1 mixture, at 55° C. in 0.1M NaCl/H₂O.

FIG. 3 shows the UV-visible absorption spectrum of a 2.4×10⁻⁵ M (on amonomeric unit basis) solution of: a) polymer 2; b) polymer 2/X1 duplex;c) polymer 2/X1/Y2 mixture, at 55° C. in 0.1M NaCl/H₂O.

FIG. 4 shows the specific optical detection of Candida albicans versusCandida dubliniensis amplicons differing by only 2 nucleotides.

FIG. 5 shows a photograph of a polymer 2—oligonucleotide duplex(solution in the left tube was colored purple-red); a polymer2—hybridized oligonucleotides triplex (solution in the middle tube wascolored yellow); and a polymer 2 with partially hybridizedoligonucleotides with two mismatches (solution in the right tube wascolored pink).

FIG. 6 shows the formation of a duplex and a triplex between polymer 2and oligonucleotides.

FIG. 7 shows the fluorescence intensity during specific hybridization.

FIG. 8 shows the fluorescence intensity during non hybridization.

FIG. 9 shows the different steps involved in the electrochemicaldetection of DNA hybridization.

FIG. 10 shows the Cyclic voltammogram of polymer 1 onto an ITO modifiedelectrode (S=50 mm²) in the case of perfect hybridization (straightline) and in the case of a control blank (dashed line) in 0.1 MNaCl/H₂O. Scan rate 100 mV/s (two successive cycles).

FIG. 11 shows the Cyclic voltammogram of polymer 2 onto an ITO modifiedelectrode (S=50 mm²) in the case of perfect hybridization (straightline) and in the case of a control blank (dashed line) in 0.1 MNaCl/H₂O. Scan rate 100 mV/s (two successive cycles).

FIG. 12 shows the covalent attachment of a DNA probe onto a conductingsurface, its subsequent hybridization with a complementary DNA strandand its revelation with a conducting polymer.

FIG. 13 shows the UV-visible absorption spectrum of a 2.4×10⁻⁵ M (on amonomeric unit basis) solution of a) polymer 2, b) polymer 2/X1 duplex,c) polymer 2/X1/Y1 triplex, d) polymer 2/X1/Y2 mixture, and e) polymer2/X1/Y3 mixture, at 55° C., in 0.1 M NaCl/H₂O. Capture probe X1: 5′CATGATTGAACCATCCACCA 3′ (SEQ ID NO: 1) and its perfect complementarytarget Y1: 3′ GTACTAACTTGGTAGGTGGT 5′ (SEQ ID NO: 2) are a DNAoligonucleotide pair specific for Candida albicans; capture probe X2: 5′CATGATTGAAGCTTCCACCA 3′ (SEQ ID NO: 3) and its perfect complementarytarget Y2: 3′ GTACTAACTTCGAAGGTGGT 5′ (SEQ ID NO: 4) are a DNAoligonucleotide pair specific for Candida dubliniensis; Y3: 3′GTACTAACTTCGTAGGTGGT 5′ (SEQ ID NO: 5) is a complementary target DNAoligonucleotide designed to have one mismatch with both the C. albicansand the C. dubliniensis capture probes.

FIG. 14 shows a cyclic voltammogram of polymer 2 (SH-carbon₂₄-DNA withpolymer 2) bound to an ITO modified electrode (50 mm²) in the case ofperfect hybridization (X1/Y1; full line) and in the case_of two controlblanks (X1/X1, dotted line and X1, dashed line) in 0.1 M NaCl/H₂O usinga scan rate of 100 mV/s.

FIG. 15 shows a cyclic voltammogram of polymer 2 (SH-carbon₂₄-DNA withpolymer 2) bound to a gold modified electrode (50 mm²) in the case ofperfect hybridization (X1/Y1; full line) and in the case of a controlblank (X1, dashed line) in 0.1 M NaCl/H₂O. The electrodes were submittedto a −0.4 V potential for 10 minutes before scanning; the scan rate usedwas 50 mV/s.

FIG. 16 shows a cyclic voltammogram of polymer 3 (SH-carbon₂₄-DNA withpolymer 3) bound to an ITO modified electrode (50 mm²) in the case ofperfect hybridization (X1/Y1; full line) and in the case of two controlblanks (X1, dashed line and silanized ITO (no DNA), dash-dotted line) in0.1 M NaCl/H₂O using a scan rate of 50 mV/s.

FIG. 17 shows the UV-visible absorption spectrum of a 2.4×10⁻⁵ M (on amonomeric unit basis) solution of: a) polymer 1/X1 duplex; b) polymer1/X1/Y1 triplex, at 55° C. in 0.1M NaCl/H₂O.

FIG. 18 shows the UV-visible absorption spectrum of a 2.4×10⁻⁵ (on amonomeric unit basis) solution of: a) polymer 4; b) X1/Y1/polymer 4triplex; c) X1/X1/polymer 4 mixture, at 25° C. in 0.1M NaCl/H₂O.

FIG. 19 shows the circular dichroism of a 1.2×10⁻⁴ M (on a monomericunit basis) solution of polymer 3/X1/Y1 triplex at 55° C. in 10 mM Trisbuffered solution containing 0.1M NaCl.

FIG. 20 shows the UV-visible spectroscopy spectrum of 7.9×10⁻⁵ Msolutions in 0.1M NaCl and TE, at 55° C. of a) polymer 2; b) duplex(X1+polymer 2); c) triplex (X1+Y1+polymer 2); d) triplex with 2mismatches (X1+5′ TGGTGGATGCATCAATCATG 3′ (SEQ ID NO: 6)), triplex with3 mismatches (X1+5′ TGGTGGATACATCAATCATG 3′ (SEQ ID NO: 7)), triplexwith 5 mismatches (X1+5′ TGGTGGAAACAACAATCATG 3′ (SEQ ID NO: 8)); e)triplex with 1 mismatch (X1+5′ TGGTGGATGCTTCAATCATG 3′ (SEQ ID NO: 9)).

FIG. 21 shows the UV-visible spectroscopy spectrum of 7.9×10⁻⁵ Msolutions in 0.1M NaCl and TE, at 55° C. of a) polymer 2; b) duplex(X1+polymer 2); c) triplex (X1+Y1+polymer 2); d) triplex with 2mismatches (X1+Y2), (X1+5′ TGGTAGATGCTTCAATCATG 3′ (SEQ ID NO: 10)),(X1+5′ TGGTGGTTGCTTCAATCATG 3′ (SEQ ID NO: 11)), (X1+5′TGGTGGATGCTTTAATCATG 3′ (SEQ ID NO: 12)), (X1+5′ TGGTGGATGCTTCATTCATG 3′(SEQ ID NO: 13)), (X1+5′ TGGTGGATGCTTCAATTATG 3′ (SEQ ID NO: 14)).

DETAILED DESCRIPTION OF THE INVENTION

Other objects and attendant features of the present invention willbecome readily appreciated, as the same becomes better understood byreference to the following detailed description of the inventiondescribed for the purpose of illustration.

In a broad sense, the invention provides novel cationic, water solublepolythiophene derivatives that produce a clearly interpretable optical(calorimetric, fluorescent or luminescent) or electrical signal, whenbound to negatively charged polymers.

The present invention provides for polymers capable of discriminatingbetween specific and non-specific hybridization of nucleic acidsdiffering by only a single nucleotide.

The present invention provides improved research tools. Morespecifically, a means for detecting nucleic acids from eucaryoticorganisms as well as prokaryotic organisms such as Bacteria and Archaea.

The present invention also provides for the development of new nucleicacid detection technology, and more specifically, new detection devicesbased on the use of the polythiophene derivatives of the presentinvention.

The present invention further provides for improved clinicaldiagnostics, that is, the detection of infectious agents, the diagnosisof genetic diseases and tools useful for use in the pharmacogenomicsfield.

The present invention further provides for improved medico-legal(forensic) diagnostics, more specifically the filiation of people andanimals, “forensic” tools and other genetic testing tools.

The present invention also provides for improved plant identification.

The present invention also provides for environmental and industrialscreening, more specifically for the detection of genetically modifiedorganisms, the detection of pathogenic agents, alimentary tracability,the identification of organisms of industrial interest (e.g. alimentary,pharmaceutical or chemical fermentation and soil decontamination).

The present invention also provides for polythiophenes having anaffinity for negatively charged polymers such as nucleic acids andglycosaminoglycans of natural or synthetic origin, allowing for thepurification of these polymers. For example, when a polythiophene iscoupled to a solid support, nucleic acids can be purified by affinityand/or ion exchange chromatography.

The present invention also provides for polythiophenes that arethermostable and autoclavable, allowing for a wide range ofapplications.

The present invention further provides methods and tools by whichnegatively charged polymers such as acidic proteins (kinesins),glycosaminoglycans (hyaluronans, heparin) and any natural or syntheticnegative polymers can be detected or blocked by binding with thepolythiophenes.

Advantageously, this novel approach is rapid, specific, sensitive, andhighly versatile, yet simple. It is based on the different electrostaticinteractions and conformational structural changes betweensingle-stranded or double-stranded negatively-charged oligonucleotidesor nucleic acid fragments, and cationic electroactive and photoactivepoly(3-alkoxy-4-methylthiophene) derivatives. It allows a single reagentassay procedure for genomic analysis and molecular diagnostics.Furthermore, the above mentioned detection methods can also be used forsolutions of nucleic acids, nucleic acids separated by gelelectrophoreses, nucleic acids fixed onto solid supports such as glassslides or plates, silicon chips or other polymers.

Synthesis

Set forth below are preferred synthesis schemes for the preparation ofthe water-soluble, cationic, electroactive and photoactivepoly(3-alkoxy-4-methylthiophene)s.

The synthesis of monomer 1 is carried out in a two-step procedure,starting from 3-bromo-4-methylthiophene (Aldrich Co.) (see Scheme 1).The first step is a nucleoplilic substitution reaction onto thethiophene ring catalyzed by CuI, as reported by El Kassmi et al. (ElKassmi et al., A.; Heraud, G.; Büchner, W.; Fache, F.; Lemaire, M. J.Mol. Catal., 72, 299, (1992)). The second step involves a quaternizationreaction between the tertiary amine and 1-bromoethane in acetonitrile.(Balanda, P. B.; Ramey, M. B.; Reynolds, J. R. Macromolecules, 32, 3970,(1999)).

Similarly, monomer 2 is prepared from3-(2-bromoethoxy)-4-methylthiophene (compound 4) and 1-methylimidazole(Aldrich Co.) (see Scheme 2). Compound 4 is prepared according to theprocedure developed by Leclerc et al. (Faïd, K.; Leclerc, M. J. Chem.Soc., Chem. Commun., 2761, (1996)). The quaternization reaction between1-methylimidazole and compound 4, provides the desired monomerimidazolium salt 2. (Lucas, P.; Mehdi, N. E.; Ho, H. A.; Belanger, D.;Breau, L. Synthesis, 9, 1253, (2000)).

Similarly, the synthesis of monomer 3 involves the conversion of3-thiopheneethanol (compound 5) into its corresponding mesylateprotected derivative (compound 6). The quaternization reaction between1-methylimidazole and compound 6, provides the desired monomerimidazolium salt 3.

The synthesis of monomer imidazolium salt 4 involves a quaternizationreaction of compound 4 with 1,2-dimethylimidazole.

A more general procedure reflecting the preparation of cationicthiophene monomers is depicted in scheme 5, wherein “m” is an integerequal to 2 or 3; “Y” is an oxygen atom or a methylene group; and R¹ is ahydrogen atom or a methyl group.

When R* is Et₃N, Y is an oxygen atom and R¹ is a methyl group, then “m”is equal to 3. When R* is 1-methylimidazole, Y is an oxygen atom and R¹is a methyl group, then “m” is equal to 2. When R* is 1-methylimidazole,Y is a methylene group and R¹ is a hydrogen atom, then “m” is equal to2. When R* is 1,2-dimethylimidazole, Y is an oxygen atom and R¹ is amethyl group, then “m” is equal to 2.

The inherent chemical and physical properties of imidazole provide for awide electrochemical window, favorable for electrochemical detection.(Bonhôte, P.; Dias, A. P.; Papageorgiou, N.; Kalyanasundaram, K.;Grätzel, M. Inorg. Chem., 35, 1168, (1996)).

All polymers, more specifically the cationic, water soluble,electroactive polymers 1, 2, 3 and 4 (Scheme 6), were synthesized byoxidative chemical polymerization of the corresponding monomers usingFeCl₃ or K₂S₂O₈ as the oxidizing agent in chloroform. This method ofpolymerization yields well-defined regio-regular3-alkoxy-4-methylthiophene polymers (1, 2 and 4) as well as anon-regioregular 3-alkylthiophene polymer (3), having an averagemolecular weight of about 5 kDa and a polydispersity index of ca. 3.(Chayer, M.; Faïd, K.; Leclerc, M. Chem. Mater., 9, 2902, (1997)). Notethat “n” can vary from 3 to about 100. The resulting polymers (usingFeCl₃ as the oxidizing agent) contain a mixture of anions such as FeCl₄⁻, Cl⁻ and Br⁻. In order to produce a cationic polymer with only onespecific counter anion (e.g. hydrophilic counter anions like Cl⁻, Br⁻,I⁻, CH₃SO₃ ⁻, etc. or hydrophobic counter anions like BF₄ ⁻, CF₃SO₃ ⁻,PF₆ ⁻, etc.), an anionic-exchange reaction is performed by dialysis orprecipitation. As expected, all resulting polymers were found to besoluble in aqueous solutions when in the presence of hydrophilic anions.

As any water-soluble cationic polyelectrolytes, the polythiophenederivatives of the present invention can make strong complexes withnegatively-charged oligomers and polymers. (Bronich, T. K.; Nguyen, H.K.; Eisenberg, A.; Kabanov, A. V. J. Am. Chem. Soc. 122, 8339, (2000)).This complexation results in the formation of complexes having specificoptical properties. For instance, at 55° C., aqueous solutions (0.1 MNaCl or 10 mM Tris buffer/0.1M NaCl) of the cationic polymer 2 areyellow (λ_(max)=397 nm). This absorption maximum at a relatively shortwavelength is related to a random coil conformation of the polythiophenederivative. (Leclerc, M. Adv. Mater. 11, 1491, (1999)). After theaddition of one equivalent (on a monomeric unit basis) of a givenoligonucleotide (20-mers), the mixture becomes red (λ_(max)=527 nm) dueto the formation of a so-called duplex. After 5 minutes of mixing in thepresence of one equivalent of a complementary oligonucleotide, thesolution becomes yellow (λ_(max)=421 nm) presumably due to the formationof a new complex (triplex) (FIGS. 1 and 5).

A schematic description of these conformational transitions for bothtypes of polyelectrolytes is given in FIG. 6.

Based on previous studies on thermochromic, solvatochromic andaffinitychromic regioregular poly(3-alkoxy-4-methylthiophene)s,(Leclerc, M. Adv. Mater. 11, 1491, (1999)) it is believed that thesecalorimetric effects are made possible due to the differentconformational structure of the conjugated polymer in the duplex (highlyconjugated, planar conformation) compared to that observed in thetriplex (less conjugated, non-planar conformation) and to a strongeraffinity of the conjugated polymer for the double-strandedoligonucleotides (nucleic acids) (1×10⁵ M⁻¹) than that measured forsingle stranded oligonucleotides (nucleic acids) (5×10⁴ M⁻¹).

In a control experiment it was demonstrated that the addition to thesolution of an oligonucleotide identical to that of the capture proberesults in no color change (FIG. 2).

In order to verify the specificity of these complexations, two pairs ofcomplementary oligonucleotides (20-mers) differing by only 1 or 2nucleotides were synthesized (Table 1) and carefully investigated. Aslight but distinct change in the UV-visible absorption spectrum isobserved in the case of the oligonucleotide target having 2 mismatches.Even with only one mismatch, it is possible to distinguish between aperfect and a non-perfect hybridization (FIG. 13). In this case, thecalorimetric difference is mainly based on different kinetics ofcomplexation, since similar yellow aqueous solutions are observed after30-60 minutes of mixing at 55° C. However, it is possible to stop thehybridization reaction after 5 minutes of mixing, by placing thesolutions at room temperature. Following these procedures, stable yellowand orange solutions are obtained (curves c and e) (FIG. 13). Thedetection limit of this colorimetric method is about 1×10¹³ molecules ofoligonucleotide (20-mers) in a total volume of 100 μL.

Very similar results have been obtained for polymer 1 (FIG. 17) andpolymer 4 (FIG. 18) and various oligonucleotides. FIG. 20 shows theUV-Visible absorbance spectrum of polymer 2 when using targetoligonucleotides ranging from 0 to 5 mismatches. FIG. 21 illustrates theUV absorbance results of polymer 2 using the target oligonucleotidealways having two mismatches at different positions. These results showthat the polymer can discriminate between perfectly matched andmismatched hybrids, independently of the nature of the mismatchednucleotide bases, and independently of the position or the length of themismatches. Moreover, it is even possible to discriminate a singlemismatch from multiple mismatches.

TABLE 1 Synthetic oligonucleotides to study the specificity ofhybridization. Oligonucleotides Oligonucleotide specific specific forCandida for Candida albicans dubliniensis X1 X2 5′ CATGATTGAACCATCCACC5′ CATGATTGAAGCTTCCACC A 3′ A 3′ (SEQ ID NO: 1) (SEQ ID NO: 3) Y1 Y23′ GTACTAACTTGGTAGGTGG 3′ GTACTAACTTCGAAGGTGG T 5′ T 5′ (SEQ ID NO: 2)(SEQ ID NO: 4) Y3 (designed to have one mismatch wit X1 and X2)3′ GTACTAACTTCGTAGGTGGT 5′ (SEQ ID NO: 5)

The concentration of a particular sequence region of DNA can beamplified by using a polymerase chain reaction (PCR), and the presentcalorimetric method can be extended to these PCR products. Indeed, theintroduction of the polymerase chain reaction (PCR) has solved theproblem of detecting small amounts of DNA and the polymers of thepresent invention can be used in the identification of PCR products. UVspectroscopic results in the absorbance range of 430-530 nm (FIG. 4)have illustrated the specific optical detection of Candida albicans andCandida dubliniensis amplicons, which differ by only 2 nucleotides, by apolymer 2/X1 duplex. Such detection was carried out in 45 minutesdirectly from a PCR product at a concentration normally generated in a100 μL PCR volume (ca. 3×10¹² copies). Experimental details on PCR andchoice of target sequences for identification of Candida are provided ina co-pending patent application (PCT/CA00/01150).

In addition, as shown in FIG. 19, circular dichroism (CD) measurementsreveal an optical activity for polymer 3 in its random coil, a bisignateCD spectrum centered at 420 nm in the triplex, characteristic of aright-handed helical orientation of the polythiophene backbone. Such aright-handed helical structure is compatible with binding of the polymerto the negatively-charged phosphate backbone of DNA. The thermalstability study by UV or CD measurements shows a different thermalstability between a duplex and a triplex, and this property could beextremely useful for more stringent washing conditions.

A fluorometric detection of oligonucleotide hybridization is alsopossible based on the difference in the fluorescence quantum yield ofthe positively-charged poly(3-alkoxy-4-methylthiophene) in the randomcoil (the isolated state) or in the aggregated state. (Chayer, M.; Faïd,K.; Leclerc, M. Chem. Mater., 9, 2902, (1997)). For instance, at 55° C.,the yellow appearance of polymer 2 is fluorescent (quantum yield of0.03) but upon addition of one equivalent of a negatively-chargedoligonucleotide, the intensity of the emission spectrum is stronglydecreased (quenched). In the case of perfect hybridization, thepolymeric triplex gives a stronger emission (FIG. 7). Upon addition ofthe same oligonucleotide, no hybridization occurs and the solution doesnot show any change in fluorescent intensity (FIG. 8). Using a laser asthe excitation source, very low limits of detection are obtained. It ispossible to detect the presence of as few as 3×10⁶ molecules of thecomplementary oligonucleotide (20-mers) in a volume of 200 μL, whichcorresponds to a concentration of 2×10⁻¹⁴ M. Moreover, by covalentlyattaching the oligonucleotide to a fluorescent-conjugated polymer, or byusing an optimized fluorescence detection scheme based on a highintensity blue diode (excitation source) and a non-dispersive,interference filter-based system, an even more sensitive and morespecific detection capability is achieved.

The electrochemical properties of polymers 1 and 2 can be used for thedetection of DNA hybridization in aqueous solutions, as shown in FIG. 9.(Youssoufi, H. K.; Garnier, F.; Srivastava, P.; Godillot, P.; Yassar, A.J. Am. Chem. Soc., 119, 7388, (1997); Garnier, F.; Youssoufi, H. K.;Srivastava, P.; Mandrand, B.; Delair, T. Synth. Metals, 100, 89,(1999)). Using the layer by layer deposition techniques (Lvov, Y.,Decher, G. & Sukhorukov, G. Assembly of thin layers by means ofsuccessive deposition of alternate layers of DNA and poly(allylamine).Macromolecules 26, 5396 (1993); Lvov, Y. M., Lu, Z., Schenkman, J. B.,Zu, X. & Rusling, J. F. Direct electrochemistry of myoglobin andcytochrome P450 in alternate layer-by-layer films with DNA and otherpolyions. J. Am. Chem. Soc. 120, 4073 (1998), the first step requiredthe binding of a capture probe composed of single-strandedoligonucleotide X1 to an ammonium-functionalized indium tin oxide (ITO)surface. (Zammatteo, N; Jeanmart, L.; Hamels, S.; Courtois, S.; Louette,P.; Hevesi, L.; Remacle, J., Anal. Biochem., 280, 143, (2000); Faïd, K.& Leclerc, M. Responsive supramolecular polythiophene assemblies J. Am.Chem. Soc. 120, 5274 (1998)). After rinsing with pure water, themodified electrode so-obtained was dipped and hybridized in the presenceof a complementary oligonucleotide Y1. The resulting electrode wasrevealed with an aqueous solution of positively-charged polymer 1 or 2(10⁻⁴ M on a monomeric basis), which provides a signal that is afunction of the amount of DNA present on the surface. As a controlexperiment, an aqueous solution of oligonucleotide X1 was added to theX1 modified ITO electrode, and then transferred into an aqueous solutionof polymer 1 or 2. In this way, these polymers serve as “masstransducers” for the oligonucleotide present in the sample.

The detection of DNA hybridization is further demonstrated by thefollowing two examples using different cationicpoly(3-alkoxy-4-methylthiophene)s (polymers 1 and 2). The so-obtainedresults are illustrated in FIGS. 10 and 11.

In both cases, the maximum anodic current is more important in the caseof perfect hybridization as compared to the blank control. In addition,a shift to a higher potential (ca. 40-50 mV) is observed when specifichybridization occurs (38 mV for polymer 4 as compared to 52 mV forpolymer 2). The higher oxidation current can be explained by thestronger affinity of the polymers for double-stranded oligonucleotides,whereas the positive shift of oxidation potential is explained by theformation of a less conjugated structure in the case of specifichybridization. This is in agreement with previous optical measurements.

An assay using a smaller electrode [S(surface)=10 mm²] has allowed thedetection of 2×10¹¹ molecules of oligonucleotide (20-mers). This verysimple electrochemical methodology is already more sensitive, by twoorders of magnitude, than the best results obtained with electrochemicalmethods using oligonucleotide-functionalized conjugate polymers.(Garnier, F.; Youssoufi, H. K.; Srivastava, P.; Mandrand, B.; Delair, T.Synth. Metals, 100, 89, (1999)). Clearly, by decreasing the size of theelectrodes and by increasing the size of the target molecules, muchlower detection limits should be obtained.

In order to further enhance the specificity of the detection, theoligonucleotide probe can be covalently attached to the polythiophenederivatives

The detection of DNA sequences can also be carried out electrochemicallyas shown below in Scheme 7.

The DNA probes can be covalently fixed to a conductive surface (FIG.12). This allows for the linking of a larger number of DNA probes to thesurface, improving the specificity and detection limit for a smallsurface.

The first step involves the modification of the conductive substrate(ITO, SnO₂, gold, doped silicon or other conductive substrate) by thecovalent attachment of a single-stranded DNA probe. The complementaryDNA strand is hybridized and the polymer is captured on the hybridizedprobe. The observed electrochemical signal is different for a hybridizedprobe (ds-DNA) versus a non-hybridized probe (ss-DNA). A linker can beused to attach the DNA probe to the conductive surface. The end groupsof the linker are such that one end readily reacts with the conductivesubstrate to form a covalent bond, whereas the other end readily reactswith an “end-modified” (SH, NH₂, COOH, etc.) DNA probe, with or withouta spacer (carbon₂₄) between the reactive function and the DNA. (Chrisey,L. A.; Lee, G. U.; O'Ferrall, C. E. Nucleic Acids Res. 24, 3031, (1996);Zammatteo, N.; Jeanmart, L.; Hamels, S.; Courtois, S.; Louefte, P.;Hevesi, L.; Remacle, J. J Anal. Biochem. 280, 143, (2000); Asanov, A.N.; Sarkisov, I. Y.; Oldham, P. B. Part of the SPIE Conference onClinical Diagnostics Systems and Technologies, 3603, 170, (1999)). Theend group of the linker that reacts with the conductive substrate'ssurface can be a silane derivative, such as an alkoxysilane, or achlorosilane. The other end group of the linker can be composed of analdehyde, a carboxylic acid, a primary amine, a succinimide ester moietyor other functional groups capable of reacting with “end-modified” DNAprobes, that is, DNA probes having specific end groups, resulting in theformation of covalent bonds with or without the help of coupling agents.

The immobilization of an oligonucleotide by means of a thiol group canbe carried out through its 3′ or 5′ terminal group. Accordingly, DNA wasimmobilized onto an ITO surface following the literature procedure forimmobilizing DNA on a glass surface. (Lenigk, R., Carles, M., Ip, N. Y.,Sucher, N. J., Langmuir, 17, 2497 (2001); Rogers, Y. H., Jiang-Baucon,P., Huang, Z. J., Bogdanov, V., Anderson, S., Boyce-Jacino, M. T., Anal.Biochem., 266, 23 (1999); Kumar, A., Larsson, O., Parodi, D., Liang, Z.,Nucl. Ac. Res., 28, e71 (2000)).

It is also possible to link “end-modified” DNA probes to various othersurfaces such as gold or other metals and metal oxides. (Flink, S.;Frank, C. J. M.; Veggel, D. N. Reinhoudt in Sensors Update, Ed. H.Baltes, W. Gopel, J. Hesse, vol. 8, Chap. 1 (2001)). Non-metallicsurfaces such as beads, glass slides, optical fibers or any othersuitable non-metallic solid support, are also possible. Theimmobilization of DNA onto a gold surface is accomplished followingknown published techniques, and the density of the attached probes canvary depending on the concentration and reaction times. (Peterson, A.W., Heaton, R. J., Georgiadis, R. M., Nucl. Ac. Res., 29, 5163 (2001);Steel, A. B., Levicky, R. L., Herne, T. M., Tarlov, M. J., Biophys. J,79, 975 (2000). The hybridization efficiency can be optimized by heatingthe substrate before adding the complementary strand. (Peterson, A. W.,Heaton, R. J., Georgiadis, R. M., Nucl. Ac. Res., 29, 5163 (2001);Peterlinz, K. A., Georgiadis, R. M., Herne, T. M., Tarlov, M. J., J. Am.Chem. Soc., 119, 3401 (1997)). (Peterson, A. W., Heaton, R. J.,Georgiadis, R. M., Nucl. Ac. Res., 29, 5163 (2001); Peterlinz, K. A.,Georgiadis, R. M., Herne, T. M., Tarlov, M. J., J. Am. Chem. Soc., 119,3401 (1997)). Polymer deposition can be optimized by dipping thesubstrate vertically into a polymer solution, and by varying the saltconcentration and the temperature of the polymer solution. The washescan be further optimized to limit the contribution of the polymer withthe non-hybridized probe.

The signal observed for a triplex should be stronger than that observedfor a duplex, with possibly a small shift towards higher potentials whenhybridization occurs. The amount of DNA is higher in the case of atriplex, which implies the presence of a higher amount of negativecharges. It is suspected that there might be two equivalents of polymer(positive charge) binding in the case of a triplex. Moreover, in thecase where only one equivalent of polymer is bound to DNA, the polymeron a non-hybridized probe (ss-DNA) is more easily washed off, ascompared to a polymer bound to DNA on a hybridized probe (ds-DNA), sinceit is less stable at high salt content or elevated temperatures. Bywashing off essentially all of the polymer from the duplex while leavingthe triplex intact, a signal coming directly from the triplex(hybridized probe+polymer) is assured. Finally, targets havingmismatches will give a different signal and possibly a lower current,due to less perfect hybridization.

The electrochemical detection of DNA hybridization is furtherdemonstrated by using polymer 2 and polymer 3, as illustrated in FIGS.14-16.

EXPERIMENTAL SECTION Example 1 General Procedure for the AromaticNucleophilic Substitution on Thiophene; Synthesis of Compound 3

Sodium hydride (0.4 g, 15 mmol) was added between 0 and 10° C. undernitrogen to a solution of N,N-diethylpropanolamine (2.0 g; 15.2 mmol) in50 mL of DME, and the resulting mixture stirred at ambient temperaturefor 20 minutes. 3-Bromo-4-methyl thiophene (2.0 g, 11.3 mmol) dissolvedin 20 mL of DME (20 mL) and CuI (1.07 g, 5.65 mmol) were then added tothe reaction mixture. The mixture was subsequently stirred overnight at95° C., while under nitrogen, diluted with methylene chloride andfiltered. The organic phase was washed three times with water, driedover MgSO₄ and evaporated. The crude product was purified bychromatography on silica gel using CH₂Cl₂ as the eluent, followed by theuse of MeOH.

Compound 3: Yield 26%; ¹H NMR (CDCl₃) δ: 1.04 (t, 6H); 1.93 (m, 2H);2.09 (s, 3H); 2.57 (m, 6H), 3.97 (t, 2H); 6.14 (d, 1H); 6.81 (d, 1H);¹³C NMR (CDCl₃) δ: 11.69; 12.50; 26.84; 46.91; 49.26; 68.19; 95.88;119.57; 129.05; 156.04; MS m/e. Calcd. For C₁₂H₂₁N₁O₁S₁; 227.1344.Found: 227.1347.

Example 2 General Procedure for Quaternization Reaction: Synthesis ofMonomer 1

1-Bromoethane (6 mL, 80.4 mmol) was added to a solution of compound 3(0.4 g, 1.8 mmol) in 60 mL of acetonitrile. The reaction mixture wasstirred at 70° C. under nitrogen for 3 days. After evaporation of theacetonitrile, the crude product was crystallized from ethyl acetate as acolorless powder.

Monomer 1: Yield 100%; m.p. 145-147° C.; ¹H NMR (CDCl₃) δ: 1.41 (t, 9H);2.06 (s, 3H); 2.31 (m, 2H); 3.57 (m, 8H), 4.12 (t, 2H); 6.24 (d, 1H);6.84 (d, 1H); ¹³C NMR (CDCl₃) δ: 7.87; 12.61; 22.55; 53.61; 54.66;65.80; 97.18; 120.28; 128.26; 154.77.

Example 3 Synthesis of Monomer 2

1-Methyl-imidazole (1.0 mL, 12.3 mmol) was added to a solution ofproduct 4 (0.54 g, 2.46 mmol) dissolved in CH₃CN (35 mL). The resultingreaction mixture was stirred at 70° C. for two days. After evaporationof the solvent, the crude product was washed twice with warm ethylacetate and twice with diethyl ether at room temperature to providemonomer 2 as a pure white solid compound.

Monomer 2: Yield 88%; 92-94° C.; ¹H NMR (CDCl₃) δ: 2.05 (s, 3H); 4.09(s, 3H); 4.38 (t, 2H); 4.90 (t, 2H), 6.27 (d, 1H); 6.82 (d, 1H); 7.63(s, 1H); 7.68 (s, 1H); 10.24 (s, 1H); ¹³C NMR (CDCl₃) δ: 12.81; 36.76;49.36; 68.01; 97.92; 120.68; 123.26; 123.30; 128.35; 137.51; 154.23.

Example 4 Synthesis of Monomer 3

Methanesulfonyl chloride (2.4 mL; 31.2 mmol) was added dropwise to asolution of 3-thiopheneethanol (2.0 g; 15.6 mmol) and triethylamine (4.3mL; 31.2 mmol), in dichloromethane (50 mL). The reaction mixture wasstirred at room-temperature for two hours. The organic phase was washedwith a NaHCO₃ solution, followed by several washings with water, and wasfinally dried with MgSO₄ and concentrated. The crude product waspurified by silica gel chromatography using CH₂Cl₂/hexane (1/1) as theeluent to yield compound 6 (59%); ¹H NMR (300 MHz, CDCl₃, 25° C., TMS):δ=2.87 (s, 3H); 3.08 (t, 2H); 4.40 (t, 2H); 6.98 (d, 1H); 7.09 (d, 1H);7.29 (dd, 1H). ¹³C NMR (300 MHz, CDCl₃, 25° C., TMS): δ=29.99; 37.19;69.59; 122.24; 125.98; 127.98; 136.44.

1-Methyl imidazole (0.4 g; 4.85 mmol) was added to a solution ofcompound 6 (0.2 g; 0.97 mmol) in toluene (20 mL). The reaction mixturewas stirred at 94° C. for two days. Following evaporation of thesolvent, the crude product was washed with warm ethyl acetate to yieldmonomer 3 as a liquid.

Monomer 3: Yield (95%); ¹H NMR (300 MHz, CDCl₃, 25° C., TMS): δ=2.72 (s,3H); 3.19 (t, 2H); 3.90 (s, 3H); 4.49 (t, 2H); 6.95 (d, 1H); 7.06 (d,1H); 7.24 (m, 2H); 7.33 (s, 1H); 9.68 (s, 1H). ¹³C NMR (300 MHz, CDCl₃,25° C., TMS): δ=30.77; 36.10; 39.67; 50.06; 122.24; 122.81; 123.01;126.46; 127.66; 136.08; 137.80.

Example 5 Synthesis of Monomer 4

The quaternization reactions were carried out following the proceduredescribed in example 3.

Monomer 4: Yield 85%; ¹H NMR (300 MHz, CDCl₃, 25° C., TMS): δ=2.02 (s,3H); 2.89 (s, 3H); 3.96 (s, 3H); 4.39 (t, 2H); 4.85 (t, 2H); 6.27 (d,1H); 6.83 (d, 1H); 7.57 (d, 1H); 7.96 (d, 1H).

Example 6 General Procedure for the Chemical Polymerization: Synthesisof Polymer 1

To a solution of iron trichloride (0.94 g, 5.8 mmol) in chloroform (23mL) under nitrogen, a solution of monomer 1 (0.487 g, 1.4 mmol) inchloroform (15 mL) was added dropwise. The mixture was stirred at roomtemperature for a period of 2 days. The reaction mixture was evaporatedto dryness and the crude product washed quickly with methanol, anddissolved in excess of acetone, and precipitated by the addition of anexcess of tetrabutylammonium chloride or tetrabutylammonium bromide. Theblack-red polymer was dissolved in methanol and dedoped by adding a fewdrops of hydrazine. The final solution was evaporated. The resultingpolymer was washed several times with a saturated solution oftetrabutylammonium chloride or tetrabutylammonium bromide in acetone,and by Soxlet extraction with acetone over a period of 6 hours, and thendried under reduced pressure to yield polymer 1 (0.32 g, 66%).

Example 7 General Procedure for Optical Detection

i) Synthetic Oligonucleotides

In a quartz UV cuvette, 100 μL (7.47×10⁻⁸ repeat units (RU) of positivecharges) of a solution of polymer 2 were added to an aqueous solution (3mL) containing either 0.1M NaCl or 10 Mm Tris buffer plus 0.1M NaCl(pH=8). The mixture was heated at 55° C. for 5 min and had a yellowappearance. 12 μL (7.47×10⁻⁸ RU of negative charges) of oligonucleotidesolution (capture probe) were then added and the resulting red solutionkept at 55° C. for an additional 5 minutes. The appropriateoligonucleotide target was added to the solution at 55° C. over 5minutes. A final yellow color is indicative of a positive result,meaning that perfect hybridization has taken place. On the other hand, ared or a red-pink color is representative of non specific or partialhybridization (two mismatches), respectively (FIGS. 1-3, respectively).

ii) PCR Products

The amplicon (double stranded 149 base pairs) from the PCR product waspre-purified by QIAQUICK DNA purification columns purchased from Qiagen(Valencia, Calif.). H₂O (90 μL) was added to a centrifuge tube followedby the addition of 6.7 μL (5.03×10⁻⁹ mol of positive charge) of asolution of polymer 2, followed by the addition of the oligonucleotidecapture probe Y1 (2 μL; 5.03 10⁻⁹ mol of negative charge) and finally,by the addition of NaCl 1M (20 μL). The resulting mixture was heated at50° C. for 10 min. The purified PCR product was freshly denatured,cooled in ice water and then added to the above solution. Thehybridization reaction was kept at 50° C. for 35 min and the colorchange observed either visually or by UV measurement (FIG. 4).

Example 8 General Procedure for Fluorescent Detection

A procedure identical to that described for optical detection wasemployed, with the exception of the use of a fluorospectrometer. Thefluorescent intensity of a “duplex” (association between a positivelycharged polymer and an oligonucleotide capture probe) was weak orinsignificant (practically zero) due to the fluorescent-quenchingproperty of the aggregated form of the polymer. When perfecthybridization does occur, the fluorescent signal becomes moresignificant (FIG. 7).

Example 9 General Procedure for Electrochemical Detection

The electrochemical test for hybridization was performed in conjunctionwith a control blank. 60 μL (1.44×10⁻⁸ mol of negative charge) ofcaptured oligonucleotide Y1 were deposited on the aminated ITO electrode(S=50 mm²) at ambient temperature for 5 min. After washing with water,60 μL (1.44×10⁻⁹ mol of negative charge) of the target oligonucleotideX1 were added and the hybridization carried out at 55° C. over 20minutes. The electrode was then cooled to room temperature over 10 minand washed twice with 0.3 M NaCl, 0.03 M NaOAc and 0.1% SDS (pH 7) andwater. 100 μL (1×10⁻⁸ mol of positive charge) of a solution of polymer 1or 2 was spread on the modified electrode for 5 min, followed by washingwith CH₃CN/H₂O (¼) and water. Cyclic voltammograms were performed inaqueous 0.1 M NaCl solutions (FIGS. 10 and 11).

Example 10 General Procedure for the Preparation of ElectrodesSubstituted with Covalent Probes

i) Covalent Attachment of DNA Probes to an ITO Electrode (Polymer 2)

ITO slides are sonicated in hexanes (10 min), methanol (10 min) andultrapure water (10 min), and are then treated with an aquaregiasolution (H₂O₂/H₂O/NH₄OH, 1/5/1) at 40° C. over a 30 minute period. Theresulting slides are rapidly washed with water, followed by sonicationin water and in acetone, then dried with air, nitrogen or argon, andheated to 110° C. for 2 to 10 min. Following this, the slides aresubmerged for three hours, while under an inert atmosphere, in anacidified ethanol solution (1 mM acetic acid in 95% ethanol) containing5% mercaptopropyltrimethoxysilane, followed by sonication in freshethanol (95%) and in ultrapure sterile water. Finally, the slides areheated at 110° C. for at least one hour, and cooled to room temperaturebefore modification with DNA.

The washes, following DNA deposition, hybridization and polymerdeposition, are carried out using an orbital shaker, unless otherwisestated. DNA attachment is carried out using a solution ofSH-carbon₂₄-DNA in sodium citrate buffer (30 mM, pH=4, 50 μL) which isdeposited onto each ITO slide, each spot forming a circle of about 1 cmin diameter. The deposition reaction is performed in a humidifiedchamber over a period of 20-24 hours. Any non-deposited DNA is drainedoff, and the slides washed with 5×SSC+0.1% Tween 20, followed bywashings with 1×SSC+0.1% Tween 20 and NaCl 0.1M.

ii) Hybridization (ITO, Polymer 2)

50 μL of RC-DNA (Y1) or of probe X1 (blank test) (25 μM in 2×SSC), aredeposited onto the DNA spot. The slides are then heated to 55° C. forabout 3 hours while in a humid atmosphere, and then cooled to roomtemperature for 15 minutes. Any non-reacted DNA is drained off and theslides washed with 2×SSC, NaCl (0.1M), with 1×SSC+0.1% Tween 20 and withNaCl (0.1M).

iii) Polymer Deposition (ITO, Polymer 2)

Polymer 2 is deposited onto the electrodes. The slides are dippedvertically over a period of 5 minutes in a solution of polymer 2 (2 mLof 10⁻⁴ M in 0.01M NaCl) at room temperature. The slides are then dippedin 2 mL of a NaCl solution (0.01M), followed by dipping in 2 mL of aCH₃CN/H₂O (¼) solution and finally, by dipping in 2 mL of a NaClsolution (0.01M).

iv) Hybridization (ITO, Polymer 3)

30 μL of Y1 (2.5 μM/NaCl 0.1M) are inserted into hybridization chamberswhich are placed onto the surface of the ITO electrode. The slides areheated for about two hours at 55° C. while in a humid atmosphere, andthen cooled to room temperature. The hybridization chambers are removedand the slides washed with a 0.1M NaCl solution and finally dried withargon.

v) Polymer Deposition (ITO, Polymer 3)

Polymer 3 is deposited onto the electrodes. A 30 μL aqueous solution ofpolymer 3 (10⁻⁴M) is inserted into hybridization chambers which are thenplaced onto the surface of the ITO electrode. The slides are heated at55° C. for 20 minutes. The hybridization chambers are_removed and theslides washed with a first portion of a 0.8M NaCl solution at 55° C.,then washed with a new portion while cooling to room temperature. Theslides are finally rinsed at room temperature with a 0.1M NaCl solution.

vi) Covalent Attachment of DNA Probes to a Gold Electrode (Polymer 2)

Gold slides are rinsed sequentially with hexanes, methanol and water.The plates are then treated with a pyranah solution (H₂O₂ 30%/H₂SO₄concentrated; 30/70) over a period of about 15 minutes. The slides arethen thoroughly washed with nanopure sterile water, and dried withargon.

DNA deposition is performed under inert atmosphere using 50 μL of asolution of SH-carbon₂₄-DNA (25 μM in 1M phosphate buffer (pH=7);K₂HPO₄+KH₂PO₄). The solution is deposited onto a 1 cm² surface, andtreated for about 16 hours under inert atmosphere. Any non-reacted DNAis then drained off, and the slides sequentially washed with H₂O, 2×SSC,and sterile water.

Polymer 2 is deposited onto the electrodes. A 50 μL solution of polymer2 (10⁻⁵ M in 0.1M NaCl) is deposited onto the DNA spot and the slidesheated to 55° C. for about 30 minutes. The slides are then washedrepeatedly with a NaCl solution (0.1M) at room temperature.

Note that the order of additions does not have to follow that describedin the above example, that is, a complementary (or a non-complementaryor with mismatches) DNA strand is added to a covalently linked DNAstrand, followed by the addition of a polymer solution. The polymersolution may be added prior to the addition of the complementary (or anon-complementary or with mismatches) DNA strand.

Example 11 The Use of the Polymers of the Present Invention to PurifyNucleic Acids and Other Such Negatively Charged Molecules

The polymers described in the present invention have high affinity fornegatively charged molecules, especially nucleic acids. In addition,they are soluble in aqueous solution and very stable under a wide rangeof temperatures. Therefore, it is possible to use these properties topurify nucleic acids and other negatively charged molecules.Chromatographic separation would involve the following steps: (1)immobilizing the polymers; (2) applying the analyte to be separated ontothe immobilized polymers under conditions such that electrostaticinteractions are possible between the analyte and the polymer; and (3)eluting the analyte by applying conditions where electrostaticinteractions between the analyte and the polymer are not favored.

Immobilization of the polymer can be achieved by covalently coupling thepolymer to a suitable solid support such as glass beads, or to beadsmade of other types of polymers. Alternatively, coupling to a solidsupport could be achieved via electrostatic interactions, such as thatdemonstrated in Example 10. In the latter case, where a captureoligonucleotide is covalently attached to a solid support, the polymerwould be expected to be eluted along with the analyte in the finalelution step since it is attached only by electrostatic bonds. Colorchanges could even be used to monitor the chromatographic process.

Conditions for binding the analyte to the polymer would preferablyinvolve solutions of low ionic charge, such as water, 0.1 M NaCl and 10mM Tris buffer/0.1 M NaCl. Different washing conditions can be appliedto remove any unwanted fractions of the analyte. pH changes couldalternatively be used to obtain conditions for binding the analyte tothe polymer.

Conditions for eluting the analyte involves solutions of high ioniccharge, such as 1.0 M NaCl solution or any other solution comprising asufficiently high counter ion concentration capable of competing for theelectrostatic interactions with the polymer. Again, pH changes couldalternatively be used to obtain conditions for eluting the analyte.

Example 12 Detection of Messenger RNA

Purified, in vitro transcribed, polyadenylated messenger RNA ofArabidopsis thaliana gene coding for NAC1, was purchased fromStratagene. Perfectly matched complementary DNA oligonucleotide N1(5′CGAGGCTTCCATCAATCTTA 3′ SEQ ID NO: 15) was synthesized byphosphoramidite chemistry on a Perkin-Elmer 391 synthesizer. UnrelatedY2 DNA oligonucleotide was obtained in the same manner. All reagents andliquid handling material coming into contact with the RNA were eithercertified RNAse-free or treated with diethylpyrocarbonate (J. Sambrookand D. W. Russel, Molecular Cloning, A Laboratory Manual, CSHL Press,2001) to inactivate RNAses. Fluorescence measurements were performed at42.5° C. on a Variant Cary Eclipse spectrofluorometer with excitationset at 420±10 nm and fluorescence emission measured at 530±5 nm,applying 1000 volts to the detector.

A duplex between polymer 2 and oligo N1 was formed by contacting8.66×10¹³ copies of oligo N1 with the equivalent amount of positivecharges of polymer 2. The reaction was carried out at room temperaturefor thirty seconds in 2 μL of water. One μl of the duplex mixture wasdiluted in 2 mL of water and put into a quartz cuvette for measurementof the initial fluorescence signal. Thereafter, a triplex was formed at42.5° C. by adding and mixing 8.66×10¹³ copies of heat treated (2minutes at 95° C.) NAC1 messenger RNA. Fluorescence of the triplex wasmeasured. A similar duplex and triplex was also made using the DNAoligonucleotide Y2, which has no significant homology with the sequenceof the NAC1 messenger RNA.

The fluorescence was significantly higher for the N1/polymer 2/NAC1triplex than for the Y2/polymer 2/NAC1 triplex, thereby showing theability of polymer 2 to distinguish between specific and nonspecifichybridization of 20 mers DNA oligonucleotides with messenger RNA.

In conclusion, a novel methodology has been developed that allows thedetection of nucleic acids by simple optical and electrochemical means.This rapid, selective, and versatile method does not require anychemical reaction on the probes or the analytes, and is based ondifferent electrostatic interactions and conformational structuralchanges between cationic poly(3-alkoxy-4-methylthiophene) derivativesand single-stranded oligonucleotides or double-stranded (hybridized)nucleic acid fragments. The present polymer-based technology is simpleand specific and provides a flexible platform for the rapid detection ofnucleic acids.

The polythiophenes are thermostable and autoclavable, hence allowing fora wide range of applications.

The terms and descriptions used herein are preferred embodiments setforth by way of illustration only, and are not intended as limitationson the many variations which those of skill in the art will recognize tobe possible in practicing the present invention, as defined in thefollowing claims.

1. A process for polymerizing a thiophene monomer, wherein saidthiophene monomer comprises a compound having the formula:

wherein: a) m is an integer ranging from 2 to 3; b) R* is a quaternaryammonium; c) Y is an oxygen atom or a methylene; and d) R¹ is a methylgroup or a hydrogen atom; wherein said process comprises: reacting saidmonomer in the presence of an oxidizing agent, resulting in theformation of a polymer of the following general formula:

wherein: a) m is an integer ranging for 2 to 3; b) n is an integerranging from 3 to 100; c) R* is a quaternary ammonium; d) Y is an oxygenatom or a methylene; and e) R¹ is a methyl group or a hydrogen atom. 2.The process of claim 1, wherein said oxidizing agent is FeCl₃ or K₂S₂O₈.3. The process of claim 1, wherein when R¹ is a methyl group, saidpolymer is regio-regular and water soluble.
 4. The process of claim 3,wherein said polymer is thermostable.
 5. A polymer obtained from theprocess of claim
 1. 6. A method for discriminating a first nucleic acidfrom a second nucleic acid, said second nucleic acid differing from saidfirst nucleic acid by at least one nucleotide, comprising the steps of:a) contacting a complementary target to said first nucleic acid with apolymer of the following general formula

wherein: a) m is an integer ranging for 2 to 3; b) n is an integerranging from 3 to 100; c) R* is a quaternary ammonium; d) Y is an oxygenatom or a methylene; and e) R¹ is a methyl group or a hydrogen atom,  toform a duplex; b) contacting said duplex with said first nucleic acid,resulting in specific hybridization; c) contacting said duplex with saidsecond nucleic acid, resulting in non-specific hybridization; and d)detecting a change in electronic charge, fluorescence or color.
 7. Themethod of claim 6, wherein said change in electronic charge,fluorescence or color is more pronounced with said specifichybridization.
 8. The method of claim 7, wherein said complementarytarget is a nucleic acid complementary to said first nucleic acid. 9.The method of claim 8, wherein said first nucleic acid and said secondnucleic acid are DNA.
 10. The method of claim 8, wherein said firstnucleic acid and said second nucleic acid are RNA.
 11. The method ofclaim 8, wherein said complementary target is a nucleic acid probe andsaid first nucleic acid and said second nucleic acid are denatured PCRamplicons.